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Toxicological information

Genetic toxicity: in vitro

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Administrative data

Endpoint:
in vitro gene mutation study in bacteria
Remarks:
Type of genotoxicity: gene mutation
Type of information:
experimental study
Adequacy of study:
key study
Study period:
2016
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2017
Report Date:
2017

Materials and methods

Test guideline
Qualifier:
according to
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
Version / remarks:
(21 Jul 1997)
Deviations:
no
GLP compliance:
yes (incl. certificate)
Remarks:
BASF SE, Experimental Toxicology and Ecology, Ludwigshafen, Germany
Type of assay:
bacterial reverse mutation assay

Test material

Reference
Name:
Unnamed
Type:
Constituent
Test material form:
solid
Details on test material:
-Batch: PO 4924335980 - ML 2005975931
- Analytical purity: 100% UVCB
- Physical state: Solid, violet
- Homogeneity: homogeneous by visual inspection
- Storage conditions: Room temperature
- The stability of the test substance under storage conditions is guaranteed until Apr 2019.
-pH of the substance: Ca. 5 (undiluted test substance, moistened with de-ionized water)
Specific details on test material used for the study:
-Batch: PO 4924335980 - ML 2005975931
- Physical state: Solid, violet
- Homogeneity: homogeneous by visual inspection
- Storage conditions: Room temperature
- The stability of the test substance under storage conditions is guaranteed until Apr 2019.
- pH of the substance: Ca. 5 (undiluted test substance, moistened with de-ionized water; determined in the lab prior to start of the GLP study)

Method

Target gene:
his+ / trp+
Species / strain
Species / strain / cell type:
S. typhimurium TA 1535, TA 1537, TA 98, TA 100 and E. coli WP2
Additional strain / cell type characteristics:
not specified
Metabolic activation:
with and without
Metabolic activation system:
phenobarbital and beta-naphthoflavone induced rat liver S9 microsomal fraction
Test concentrations with justification for top dose:
1st Experiment (Standard plate test with and without S9 mix): 0; 33; 100; 333; 1000; 3925 and 7850 μg/plate
2nd Experiment (Preincubation test with and without S9 mix): 10 μg - 3925 μg/plate

The dose refers to the test material, not to the amount of the pure target substance.

Vehicle / solvent:
- Vehicle(s)/solvent(s) used: DMSO
- Justification for choice of solvent/vehicle: Due to the limited solubility of the test substance in ultrapure water, ethanol was used as vehicle, which had been demonstrated to be suitable in bacterial reverse mutation tests and for which historical control data are available.
Controls
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
other: 2-aminoanthracene (2-AA), N-methyl-N'-nitro-N-nitrosoguanidine (MNNG), 4-nitro-o-phenylenediamine (NOPD), 9-aminoacridine (AAC), 4-nitroquinoline-N-oxide (4-NQO)
Details on test system and experimental conditions:
METHOD OF APPLICATION: in agar (plate incorporation); preincubation

Standard plate test:
- Exposure duration: 48 – 72 hours

Preincubation test:
- Preincubation period: 20 min
- Exposure duration: 48 – 72 hours


NUMBER OF REPLICATIONS: 3


DETERMINATION OF CYTOTOXICITY
- Method: decrease in the number of revertants, clearing or diminution of the background lawn, reduction in the titer


OTHER:
Titer determination: The titer was determined only in the experimental parts with S9 mix both for the negative controls (vehicle only) and for the two highest doses in all experiments.
The concentrations have been adusted for the low content in the sample.

Positive controls:
With S9 mix: 2-aminoanthracene (2-AA), 2.5 μg/plate, dissolved in DMSO / TA 1535, TA 100, TA 1537, TA 98; 60 μg/plate, dissolved in DMSO / Escherichia coli WP2 uvrA
Without S9 mix: N-methyl-N'-nitro-N-nitrosoguanidine (MNNG), 5 μg/plate, dissolved in DMSO / TA 1535, TA 100; 4-nitro-o-phenylenediamine (NOPD), 10 μg/plate, dissolved in DMSO / TA 98; 9-aminoacridine (AAC), 100 μg/plate, dissolved in DMSO / TA 1537; 4-nitroquinoline-N-oxide (4-NQO) (SIGMA, N-8141), 5 μg/plate, dissolved in DMSO / E. coli WP2 uvrA
Evaluation criteria:
Acceptance criteria:
Generally, the experiment is considered valid if the following criteria are met:
• The number of revertant colonies in the negative controls was within the range of the historical negative control data for each tester strain.
• The sterility controls revealed no indication of bacterial contamination.
• The positive control substances both with and without S9 mix induced a distinct increase in the number of revertant colonies within the range of the historical positive control data or above.
• Fresh bacterial culture containing approximately 10^9 cells per mL were used. For approval the titer of viable bacteria was ≥ 10^8 colonies per mL.

Assessment criteria:
The test substance is considered positive in this assay if the following criteria are met:
• A dose-related and reproducible increase in the number of revertant colonies, i.e. about doubling of the spontaneous mutation rate in at least one tester strain either without S9 mix or after adding a metabolizing system.

A test substance is generally considered non-mutagenic in this test if:
• The number of revertants for all tester strains were within the historical negative control range under all experimental conditions in at least two experiments carried out independently of each other.

Cytotoxicity criteria:
Toxicity detected by a
• decrease in the number of revertants (factor ≤ 0.6)
• clearing or diminution of the background lawn (= reduced his- or trp- background growth) was recorded for all test groups both with and without S9 mix in all experiments and indicated in the tables.

In low dose group, single values with a factor ≤ 0.6 were not considered as in indicator of toxicity.

Results and discussion

Test resultsopen allclose all
Species / strain:
E. coli WP2 uvr A
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
not examined
Positive controls validity:
valid
Species / strain:
S. typhimurium TA 1535
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
not examined
Positive controls validity:
valid
Species / strain:
S. typhimurium TA 1537
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
not examined
Positive controls validity:
valid
Species / strain:
S. typhimurium TA 100
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
not examined
Positive controls validity:
valid
Species / strain:
S. typhimurium TA 98
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
not examined
Positive controls validity:
valid
Additional information on results:
TEST-SPECIFIC CONFOUNDING FACTORS
- Precipitation: Precipitation of the test substance was found in the standard plate test from about 1000 μg/plate onward with and without S9 mix.

COMPARISON WITH HISTORICAL CONTROL DATA: yes

ADDITIONAL INFORMATION ON CYTOTOXICITY: A bacteriotoxic effect was observed depending on the strain and test conditions from about 1000 μg/plate onward. For details it is refered to table 1.

Any other information on results incl. tables

Table 2: Standard plate test, without S9 mix

Strain Test group Dose
(μg/plate)
Mean
revertants
per plate
Standard
deviation
Factor Individual revertant colony
counts
TA 1535 DMSO - 10.0 1.7 - 8, 11, 11
Test item 33 10.0 1.7 1.0 9, 9, 12
100 14.3 4.2 1.4 13, 19, 11
333 13.3 2.1 1.3 14, 15, 11
1000 12.0 1.0 1.2 12 P, 11 P, 13 P
3925 7.0 4.4 0.7 9 P, 2 P, 10 P
7850 1.7 1.2 0.2 3 P, 1 P, 1 P
MNNG 5.0 5616.0 433.2 561.6 5920, 5808, 5120
TA 100 DMSO - 105.0 5.6 - 106, 99, 110
Test item 33 112.3 12.5 1.1 100, 112, 125
100 113.7 8.7 1.1 121, 116, 104
333 77.3 11.0 0.7 90, 72, 70
1000 83.0 11.5 0.8 82 P, 95 P, 72 P
3925 70.0 18.0 0.7 65 P, 55 P, 90 P
7850 38.3 7.6 0.4 47 P, 35 P, 33 P
  MNNG 5.0 3868.7 245.2 36.8 4071, 3939, 3596
TA 1537 DMSO - 7.7 0.6 - 8, 7, 8
Test item 33 9.0 1.0 1.2 10, 9, 8
100 9.7 2.1 1.3 8, 9, 12
333 7.7 2.9 1.0 6, 11, 6
1000 6.0 2.6 0.8 7 P, 8 P, 3 P
3925 3.7 1.2 0.5 5 P, 3 P, 3 P
7850 1.0 1.0 0.1 1 P, 2 P, 0 P
  AAC 100 695.7 310.2 90.7 409, 653, 1025
TA 98 DMSO - 23.3 4.2 - 28, 20, 22
Test item 33 22.7 4.0 1.0 19, 27, 22
100 25.0 6.2 1.1 27, 30, 18
333 19.0 5.3 0.8 17, 15, 25
1000 18.7 3.1 0.8 18 P, 22 P, 16 P
3925 13.3 3.8 0.6 9 P, 15 P, 16 P
7850 8.0 1.0 0.3 9 P, 8 P, 7 P
  NOPD 10 954.7 54.8 40.9 923, 923, 1018
E. coli DMSO - 28.3 3.8 - 24, 31, 30
Test item 33 21.7 3.8 0.8 19, 26, 20
100 22.7 3.5 0.8 19, 23, 26
333 21.7 7.5 0.8 13, 26, 26
1000 23.3 9.5 0.8 14 P, 33 P, 23 P
3925 9.0 1.0 0.3 9 P, 10 P, 8 P
7850 11.7 0.6 0.4 12 P, 11 P, 12 P
  4-NQO 5 1454.0 43.5 51.3 1439, 1503, 1420

Table 3, Standard plate test with metabolic activation

Strain Test group Dose
(μg/plate)
Mean
revertants
per plate
Standard
deviation
Factor Individual revertant colony
counts
TA 1535 DMSO - 10.3 0.6 - 10, 11, 10
Test item 33 11.7 3.5 1.1 12, 8, 15
100 10.3 2.3 1.0 13, 9, 9
333 9.3 1.5 0.9 8, 11, 9
1000 7.3 1.5 0.7 6 P, 9 P, 7 P
3925 4.3 1.5 0.4 3 P, 6 P, 4 P
7850 3.7 0.6 0.4 4 P, 3 P, 4 P
  2-AA 2.5 193.0 22.5 18.7 205, 207, 167
TA 100 DMSO - 114.7 2.5 - 117, 112, 115
Test item 33 116.3 11.2 1.0 112, 108, 129
100 114.3 5.1 1.0 110, 113, 120
333 107.3 3.2 0.9 111, 106, 105
1000 86.0 8.2 0.8 95 P, 79 P, 84 P
3925 80.3 19.5 0.7 58 P, 89 P, 94 P
7850 80.3 8.1 0.7 71 P, 86 P, 84 P
  2-AA 2.5 1732.0 88.1 15.1 1748, 1637, 1811
TA 1537 DMSO - 8.7 1.2 - 8, 8, 10
Test item 33 10.3 2.3 1.2 9, 13, 9
100 8.0 2.6 0.9 11, 7, 6
333 7.3 1.2 0.8 6, 8, 8
1000 7.7 2.1 0.9 7 P, 10 P, 6 P
3925 4.3 1.5 0.5 4 P, 6 P, 3 P
7850 2.0 1.7 0.2 1 P, 1 P, 4 P
  2-AA 2.5 144.0 19.5 16.6 124, 145, 163
TA 98 DMSO - 28.0 2.6 - 25, 30, 29
Test item 33 24.3 6.1 0.9 31, 19, 23
100 23.3 3.2 0.8 21, 22, 27
333 31.0 7.2 1.1 29, 25, 39
1000 23.3 5.7 0.8 25 P, 17 P, 28 P
3925 15.3 6.0 0.5 21 P, 9 P, 16 P
7850 7.0 1.0 0.3 8 P, 6 P, 7 P
  2-AA 2.5 1388.7 140.7 49.6 1301, 1551, 1314
E. coli DMSO - 24.7 4.5 - 20, 29, 25
Test item 33 30.7 4.2 1.2 34, 26, 32
100 26.0 1.7 1.1 25, 25, 28
333 27.0 7.5 1.1 26, 35, 20
1000 21.7 6.7 0.9 26 P, 25 P, 14 P
3925 19.0 3.0 0.8 22 P, 16 P, 19 P
7850 12.7 3.2 0.5 15 P, 9 P, 14 P
  2-AA 60 133.7 10.1 5.4 139, 122, 140

Applicant's summary and conclusion

Conclusions:
The substance is not mutagenic in bacteria.